Q5 Blood Direct 2X Master Mix can amplify a wide variety of targets direct from dried blood spots or up to 30% whole human blood, skipping DNA purification. The master mix includes Q5 Hot Start High-Fidelity DNA Polymerase, a thermostable DNA polymerase, with 3′→ 5′ exonuclease activity, fused to a processivity-enhancing Sso7d domain and dNTPs in an optimized buffer with increased resistance to inhibitors in blood, anti-coagulants, and chemicals on filter papers. It is capable of amplifying products up to 7.5 kb from human whole blood cells preserved with sodium EDTA, potassium EDTA, sodium citrate and sodium heparin as well samples stored on common preservative filter papers.
Q5 Hot Start High-Fidelity DNA Polymerase offers robust DNA amplification with low error rates. The addition of an aptamer-based inhibitor allows for room temperature reaction setup.
It is recommended to use NEB’s Tm Calculator to determine the appropriate annealing temperature for each primer pair and polymerase/buffer of interest. Unlike other calculators, the NEB Tm Calculator takes buffer components that affect melting temperatures into consideration when calculating the best annealing temperature. Other online calculators may underestimate the best Q5 annealing temperature.
Figure 1: Q5 Blood Direct 2X Master Mix enables robust amplification direct from whole or dried blood
PCR was performed using Q5 Blood Direct 2X Master Mix under standard recommended conditions with 35 cycles of amplification. Yield and purity were quantitated by microfluidic LabChip® analysis and are indicated by dot size and color, respectively, with a large, dark green dot representing the strongest performance. A. Amplification of a variety of human genomic amplicons, 0.3 to 7.5 kb in length, from 10% EDTA-preserved human whole blood. Results are shown as both a virtual gel (top) and corresponding dot plot (bottom). Q5 Blood Direct 2X Master Mix performs well across a broad range of amplicon sizes. B. Amplification of a 604 bp human genomic amplicon from whole blood (top) or blood dried on filters (bottom). Human whole blood comprised 5-30% of the total reaction volume (50 µl) as indicated. Untreated 1 mm punches from dried blood spots were added directly to 25 µl reactions (one punch per reaction), even where pre-treatment of the punch was recommended by the manufacturer. Q5 Blood Direct 2X Master Mix shows broad tolerance to varying blood volumes, preservatives and punch types.
Figure 2: Q5 Blood Direct 2X Master Mix offers strong performance on the widest variety of targets
PCR was performed with human amplicons varying in (A) GC content (23-77%) and (B) length (2 to 7 kb), using Q5 Blood Direct 2X Master Mix and several other commercially available blood direct polymerases. 10% EDTA-preserved human whole blood was used as starting material input. 50 µl reactions were cycled according to manufacturers’ recommendations. Yield and purity were quantitated by microfluidic LabChip® analysis and are indicated by dot size and color, respectively. Q5 Blood Direct 2X Master Mix shows consistently strong performance for the broadest range of targets.
Figure 3: Consistent performance across filter types, blood preservatives and reaction volumes
PCR was performed with a 604 bp human genomic amplicon from dried blood using Q5 Blood Direct 2X Master Mix under standard recommended conditions. Whatman 903 or FTA Elute paper was spotted with blood preserved with various anticoagulants. Punches from these dried blood spots were added direct to 20, 25 or 50 µl reactions, without pre-treatment. Amplification was robust regardless of volume size, anticoagulant, or filter type.
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This product is licensed for research and commercial use from Bio-Rad Laboratories, Inc., under U.S. Pat. Nos. 6,627,424, 7,541,170, 7,670,808, 7,666,645, and corresponding patents in other countries. No rights are granted for use of the product for Digital PCR or real-time PCR applications, with the exception of quantification in Next Generation Sequencing workflows
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